A Review of Natural and Engineered Enzymes Involved in Bioethanol Production

University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2016 A Review of natural and engineered enzymes involved in bioethanol production Ines Cuesta Urena Follow this and additional works at: https://scholarworks.umt.edu/etd Part of the Biochemistry Commons, and the Biotechnology Commons Let us know how access to this document benefits ou.y Recommended Citation Cuesta Urena, Ines, "A Review of natural and engineered enzymes involved in bioethanol production" (2016). Graduate Student Theses, Dissertations, & Professional Papers. 4562. https://scholarworks.umt.edu/etd/4562 This Professional Paper is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. A REVIEW OF NATURAL AND ENGINEERED ENZYMES INVOLVED IN BIOETHANOL PRODUCTION By Inés Cuesta Ureña Bachelor’s of Science in [Biology], University of Barcelona, Barcelona, Spain, 2011 Professional Paper presented in partial fulfillment of the requirements for the degree of Master of Interdisciplinary Studies The University of Montana Missoula, MT December 2015 Approved by: Sandy Ross, Dean of The Graduate School Graduate School Michael Ceballos, Chair University of Minnesota Morris, Division of Science and Mathematics Sandy Ross, PhD Department of Chemistry Klara Briknarova, PhD Department of Chemistry Biswarup Mukhopadhyay, PhD VirginiaTech, Department of Biochemistry Cuesta Ureña, Inés, M.A., fall 2015 Interdisciplinary Studies A Review of natural and engineered enzymes involved in bioethanol production. Alternative petroleum-derived fuels, such as biofuels, is another form to decrease the dependence of non-renewable energy. The most promising alternative energy is cellulosic ethanol because of the abundance of cellulose and the overall lack of concern for the food- versus-fuel dilemma. In order to produce ethanol from cellulosic materials, pretreatment is required to “open” the lignocellulosic matrix and make cellulose more susceptible to enzymatic degradation. Enzymatic hydrolysis of lignocellulose is an important area of research due to the absence of negative effects in downstream processes in comparison with acid hydrolysis. Both natural enzymes and engineered enzymes can be used in the process of ethanol production. Natural enzymes are found either individually or as a part of a complex known as cellulosome. Such complexes are the focus of many studies due to the efficiency in the degradation of cellulose. Research in enzymatic engineering is being done in order to mimic these natural systems. Engineered individual enzymes are also used to improve the properties of the enzymes found in nature. Enzymes can be engineered by rational design or directed evolution. Directed evolution is the most efficient technology, since it only requires the knowledge of protein sequences. However, this approach also possesses some limitations. A combination of both methods or a “semi-rational” approach is perhaps the best option to develop higher performance lignocellulolytic enzymes. Many advances regarding engineering of lignocellulolytic enzymes have been made in the last past years. Further research, however, is required in the development of enzymes systems and enzyme industrial testing to establish cellulosic bioethanol as main substitute for petroleum-derived fuel energy. Chairperson or Co-Chairperson: Ceballos, Michael ii INTRODUCTION The interest in alternative energies has increased in the past years due to the unsustainable use of natural resources and their growing demand. Biofuels, which can be made from lipid-rich feedstocks (biodiesel) or carbohydrate-rich feedstocks (bioethanol), is one such form energy. Biofuels are considered renewable and, in many cases, sustainable and environmentally-friendly. Although bioethanol possesses numerous advantages, there are still some limitations in commercial production that prevent it from being cost competitive and the liquid fuel of choice. These limitations include: the use of land and water resources to produce corn- and sugarcane- based bioethanol (i.e., first generation biofuels) that competes with food crop production and the inefficient conversion of feedstock molecular substrates to fermentable sugars in cellulosic ethanol (i.e., second generation bioethanol). Current research is focused on overcoming these limitations. Since cellulose is the most abundant polymer on Earth and first generation bioethanol production creates a food-versus-fuel dilemma, cellulosic ethanol may be the most promising alternative liquid fuel option if production process bottlenecks can be resolved. Pretreatment of cellulosic feedstock is typically required to expand or “loosen up” the lignocellulosic matrix. At the molecular level this entails: breaking hydrogen bonds between lignin and holocellulose; disrupting hydrogen bonding within holocellulose; lysing covalent bonds that stabilize lignin; and, destabilizing the crystalline of cellulose to render it more susceptible to degradation by sugar reducing enzymes. Depolymerization of molecular substrates (e.g., cellulose) from pretreated lignocellulosic materials to generate simpler fermentable, sugars (e.g., monosaccharides) is often achieved by one of two different sugar reduction processes: enzymatic hydrolysis; or, acid hydrolysis. Acid hydrolysis is mainly performed via sulfuric acid; however, due to negative effects in downstream processes, the use of this technology is limited. Using hydrolytic enzymes from microorganisms (i.e., bacteria and fungi) as a substitute for chemical approaches is technology continuously in development and an area of intense research. Enzymatic degradation of lignocellulose is performed using a variety of enzymes in specific ratios or sequences so that biomass deconstruction and generation of fermentable sugar may be achieved in an efficient manner. Multi-enzyme systems are designed to optimize synergistic interactions between different classes of enzymes during this process. In general, enzymes used in this process are referred to as “lignocellulolytic enzymes”. Most are derived from natural systems and each class of enzymes possesses unique features including structural-functional modularity. Optimization of enzyme-mediated processes is particularly important if bioethanol is to become economically viable. Either enzymes found in nature or engineered enzymes can be utilized in industrial-scale bioethanol production processes. Attempts to mimic the activity of naturally- occurring extracellular macromolecular complexes called cellulosomes have been undertaken, 1 because of the efficiency by which cellulosomes degrade cellulosic biomass in nature. This has spawned the development of enzyme cocktails and engineered platform systems. The goal of this article is to provide a review of the enzymes found in nature as well as engineered enzymes that are used in the production of bioethanol with focus on their respective modes of action and a description of the most important multienzyme systems found in nature are discussed, including the cellulosome of C. thermocellum. NATURAL ENZYMES USED TO CONVERT FEEDSTOCK TO SUBSTRATE Mode of Action of Primary Lignocellulolytic Enzymes To use cellulosic material as feedstock for bioethanol production, chipped or ground biomass is typically pretreated to facilitate enzyme access to long chain carbohydrates (e.g., cellulose), which are the macromolecules that are reduced to fermentable sugar for conversion to ethanol. Given the heterogeneous nature of lignocellulose, it is highly recalcitrant even with pretreatment. Numerous methods have been developed for degrading lignocellulose to expose polysaccharides. Likewise, numerous approaches have been developed to reduce these macromolecular substrates. Those approaches that rely on enzymes produced by microorganisms can be highly efficient. Both multi-domain enzymes and enzyme complexes (e.g., mini-cellulosomes) have been applied. One useful feature of many lignocellulolytic enzymes (and their complexes) is innate modularity. In addition to a catalytic core region, many cellulolytic enzymes possess non-catalytic domains. Two notable domains include carbohydrate-binding modules (CBMs) and dockerin domains. CBMs facilitate interactions between enzymes and their respective carbohydrate substrates (Tomme et al., 1988; Tomme et al., 1998; Boraston et al., 1999; Gilbert et al., 2013). Various studies have demonstrated CBMs enhance enzymatic activity against recalcitrant substrates (Black et al., 1996; Bolam et al., 1998; Carrard et al., 2000; Mello and Polikarpov, 2014). Dockerin domains on cellulolytic enzymes from some species of microorganisms mediate cohesin–dockerin interactions, associating the enzymes with larger macromolecular complexes. These complexes, or cellulosomes, are found naturally at the cell membrane-cell wall structure of many cellulolytic microorganisms (see Fontes and Gilbert, 2010). Lignocellulolytic enzymes may be generally categorized as: cellulases, hemicellulases, ligninolytic enzymes and pectinases. This section provides a review of primary lignocellulolytic enzymes and their respective functions within natural cellulosomes. 2 Cellulases Cellulases are glycosyl or glycoside hydrolases (GHs) that catalyze

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